Electrospray ionization-mass spectrometry (ESI-MS) has become a routine tool in proteomic studies, primarily due to its high sensitivity, broad dynamic range, and versatility for online coupling with capillary high performance liquid chromatography (HPLC)1-3. High-resolution separation prior to MS detection allows complex mixtures to be characterized by extending both the dynamic range and detection level achievable in the analysis. Capillary LC, using 75-150 μm i.d. reversed-phase columns, offers the advantages of high resolving power, high mass sensitivity, and low sample and mobile phase consumption, and hence are widely used today. However, even with such columns, LC-MS analysis of very low quantity samples (e.g., cells from small tissue samples obtained using laser capture microdissection4) can still be problematic. More sensitive proteomic analysis methods are necessary to tackle many challenging biological problems.
For a given injected sample amount, narrow-bore columns result in reduced chromatographic band dilution, the analytes being eluted in a smaller volume at a higher concentration. In addition, the volumetric flow rate is an important parameter influencing ESI sensitivity5-10. Low flow rates resulting from the narrow bore columns lead to smaller electrospray droplet sizes, thus enhancing analyte ionization efficiency and reducing the effect of ion suppression, all leading to higher sensitivity. Additionally, the electrospray emitter attached to such a low flow rate column can be placed nearer to the MS inlet than in comparable configurations, which improves the sampling efficiency at low flow rates. NanoESI, at flow rates of <30 nL/min, will, thus, significantly increase the MS response compared to conventional flow rates (>300 nL/min)5,6,11. On the other hand, packing narrow-bore (<20 μm i.d.) columns with conventional microparticles can be technically difficult because the decreased ratio of column i.d. to particle size induces more frequent column clogging, and packing microparticles into narrow-bore (<20 μm i.d.) columns requires ultrahigh packing pressure (usually >10,000 psi) and special instrumentation. Generally, the ratio of column i.d. to particle size should be greater than 10 to pack dense columns reproducibly. Recently, the preparation of 10 μm i.d. columns packed with 1.0 μm non-porous particles at extremely high pressure has been reported12. The back pressure of a 30 cm long column can reach as high as 100,000 psi at the optimum linear velocity of 0.4 cm/s. Monolithic capillary columns are increasingly considered as a viable alternative to microparticle-packed columns because of their moderate back pressure and high resolving power6,8,13-16. It was recently demonstrated that low-attomole sensitivity can be achieved at a flow rate of 20 nL/min using a 20 μm i.d. PS-DVB monolithic column6. Even more recently, others have reported on the preparation of 20 and 10 μm i.d. silica-based monolithic columns8,16, demonstrating sensitive and quantitative proteomic analyses at the very low flow rate of 10 nL/min16. However, in all these cases, preparation of the monolithic columns was difficult, in part due to the increased surface area to column i.d. ratio.
Given the excellent performance of open tubular capillary gas chromatography (GC), researchers have for many years tried to implement such columns for LC. It was recognized early on49 that very narrow bore columns of 5-10 μm i.d. were necessary for open tubular LC, in order to overcome band broadening due to the laminar flow in the capillary tube. Approaches of coating the capillary tubing using silicone17 or chemical modification of etched surfaces18 were first developed to prepare open tubular capillary LC columns. However, such columns provided low retention and low sample loading capacity even for small molecules, let alone for complex biological samples.
Porous layer open tube (PLOT) columns were introduced in 1960s to increase the sample loading capacity of the GC columns19. Although efforts have been made in the last 20 years to prepare PLOT capillary LC columns20-23, success has been limited due in part to the following: 1) lack of a sensitive, universal, small dead volume detector24; 2) lack of ability to generate effective gradient elution at very low flow rates; and 3) difficulties in the preparation of capillary columns with a uniform stationary layer reproducibly. ESI-MS has proven to be an ideal sensitive detector with zero dead volume, and current HPLC pumps can provide stable flow rate at low nL/min level after accurate splitting. The remaining problem is to prepare and implement high efficiency LC PLOT columns, a major challenge being to cast a suitably uniform porous layer on the column to provide sufficient retention and sample loading capacity. Several methods have been developed to realize a retentive layer suitable for increasing the surface area and phase ratio, e.g., static25, dynamic26,27, and precipitation coating28. To simplify the preparation process, a method of laying down a porous siliceous layer in a single step via a sol-gel process was described29. Methods of preparing gold nanoparticle-coated PLOT columns have also been described30,31. However, these and other attempts32-36 have not been sufficiently successful to permit commercial level development of PLOT capillary LC columns and their use, e.g., in ESI-MS.